Use of mesenchymal stem cells to enhance or restore fertility potential: a systematic review of available experimental strategies

Abstract STUDY QUESTION To what extent does regenerative medicine with stem cell therapy help to address infertility issues for future clinical application? SUMMARY ANSWER Regenerative medicine using different stem cell sources is yielding promising results in terms of protecting the ovarian reserve from damage and senescence, and improving fertility potential in various preclinical settings. WHAT IS KNOWN ALREADY Regenerative medicine using stem cell therapy is emerging as a potential strategy to address a number of issues in the field of human reproduction. Indeed, different types of adult and fetal mesenchymal stem cells (MSCs) have been tested with promising results, owing to their ability to differentiate into different tissue lineages, move toward specific injured sites (homing), and generate a secretome with wound-healing, proangiogenic, and antioxidant capacities. STUDY DESIGN, SIZE, DURATION Guided by the checklist for preferred reporting items for systematic reviews and meta-analyses, we retrieved relevant studies from PubMed, Medline, and Embase databases until June 2023 using the following keywords: ‘mesenchymal stem cells’ AND ‘ovarian follicles’ OR ‘ovarian tissue culture’ OR ‘ovarian follicle culture’ OR ‘cumulus oocyte complex’. Only peer-reviewed published articles written in English were included. PARTICIPANTS/MATERIALS, SETTING, METHODS The primary outcome for the experimental strategies was evaluation of the ovarian reserve, with a focus on follicle survival, number, and growth. Secondary outcomes involved analyses of other parameters associated with the follicle pool, such as hormones and growth factors, ovarian tissue viability markers including oxidative stress levels, oocyte growth and maturation rates, and of course pregnancy outcomes. MAIN RESULTS AND THE ROLE OF CHANCE Preclinical studies exploring MSCs from different animal origins and tissue sources in specific conditions were selected (n = 112), including: in vitro culture of granulosa cells, ovarian tissue and isolated ovarian follicles; ovarian tissue transplantation; and systemic or intraovarian injection after gonadotoxic or age-related follicle pool decline. Protecting the ovarian reserve from aging and gonadotoxic damage has been widely tested in vitro and in vivo using murine models and is now yielding initial data in the first ever case series of patients with premature ovarian insufficiency. Use of MSCs as feeder cells in ovarian tissue culture was found to improve follicle outcomes and oocyte competence, bringing us one step closer to future clinical application. MSCs also have proved effective at boosting revascularization in the transplantation site when grafting ovarian tissue in experimental animal models. LIMITATIONS, REASONS FOR CAUTION While preclinical results look promising in terms of protecting the ovarian reserve in different experimental models (especially those in vitro using various mammal experimental models and in vivo using murine models), there is still a lot of work to do before this approach can be considered safe and successfully implemented in a clinical setting. WIDER IMPLICATIONS OF THE FINDINGS All gathered data on the one hand show that regenerative medicine techniques are quickly gaining ground among innovative techniques being developed for future clinical application in the field of reproductive medicine. After proving MSC effectiveness in preclinical settings, there is still a lot of work to do before MSCs can be safely and effectively used in different clinical applications. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0077.14, FNRS-CDR J.0063.20, and grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, and the Fondation St Luc. None of the authors have any competing interest to disclose. REGISTRATION NUMBER N/A.


Introduction
Regenerative medicine is emerging as a potential tool to manage various pathological conditions that have no current treatment.There is particular interest in reproductive medicine, since the number of ovarian follicles, which are the main functional units of the ovary and responsible for female fertility, is finite at birth and continues to fall during the reproductive lifespan, with no ability to regenerate (Dolmans et al., 2021a).Moreover, follicles, and especially the oocytes contained within, are characterized by specific damage repair mechanisms, as their key role is to convey undamaged genetic information to future offspring (Maidarti et al., 2020).This makes follicles particularly vulnerable to potentially damaging effects of various stimuli, resulting in reduced fertility potential and premature depletion of the ovarian reserve.
Regenerative medicine techniques are based on use of stem cells, which are defined as cells originating from a multicellular organism that are capable of giving rise to infinitely more cells of the same type (self-renewal), as well as other cell types, by differentiation (potency).They represent populations of non-specialized cells that have the potential to differentiate into specialized cellular subtypes (Weissman, 2000).Stem cells can be classified according to their origin into embryonic, fetal, adult, and induced pluripotent stem cells (Takahashi and Yamanaka, 2006;Bacakova et al., 2018).
The present review will focus exclusively on fetal and adult stem cells since their use as therapeutic tools is not contentious and is actually considered the most promising for regenerative medicine and tissue engineering purposes.Fetal stem cells can be isolated from various surplus fetal tissues, such as amnion, chorion, amniotic fluid and the umbilical cord, and show greater multilineage differentiation capacity than adult stem cells.Adult, or somatic, stem cells are located in all organs and tissues to varying degrees, with the function of maintaining and repairing them (Ding et al., 2011).Most of them are multipotent, with cell lineage-specific restrictions, or oligo/unipotent, also known as progenitor cells (Melchiorri et al., 2016).Some, like MSCs, are even able to express multipotency toward other cell lineages in specific conditions (Bacakova et al., 2018).
The impact of MSCs appears to depend on their capacity to secrete a diversity of cytokines, chemokines, and growth factors.Some of these secreted factors play a crucial role in controlling cell proliferation and apoptosis rates, thereby promoting regeneration of injured tissues (Wang et al., 2011).MSCs also exert a modulatory effect on the immune system (Wei et al., 2013), suppressing excessive responses by macrophages, dendritic cells, and natural killer cells through cell-to-cell contact and release of soluble immunosuppressive factors (Uccelli et al., 2008).They also possess homing properties, namely the capacity to directionally migrate to distant damaged organs/tissues in response to signaling molecules (Moser and Loetscher, 2001).These abilities have fostered growing interest in the field of regenerative medicine based on the idea that MSC infusions or localized therapy may well aid organ and tissue repair.
Increasing evidence of the potential of MSCs to treat different diseases is currently being gathered to facilitate their transition from bench to bedside.Different disease models are being tested and numerous clinical trials are ongoing (Rodr� ıguez-Fuentes et al., 2021).While significant progress has been made, stem cell therapy is still several steps away from use in clinical practice.One of the main issues is standardizing the methodology to isolate, characterize, and expand MSCs before their clinical application.This is not always easy, as it may involve different MSC subpopulations that could later show heterogeneous behavior in vitro (Baer and Geiger, 2012).Such heterogeneity is contingent

WHAT DOES THIS MEAN FOR PATIENTS?
'Regenerative medicine' describes a potential clinical approach for managing various pathological conditions (i.e.diseases or injury) that have no current treatment options, including many in the field of human reproduction.The techniques involved are based on the use of mesenchymal stem cells (MSCs), which are non-specialized cells that can give rise to infinitely more cells of the same type, as well as other cell types.Stem cells have the potential to regenerate and produce signals that promote wound healing (i.e.tissue regeneration) in different organs.For this reason, various experimental strategies are under development to exploit the ability of stem cells to protect or restore fertility.More specifically, stem cells may help protect the ovary (and the follicles/eggs it contains) against different types of injury, caused either by the aging process or use of chemotherapy after a cancer diagnosis.Both conditions significantly decrease a woman's fertility and chance of pregnancy.Would MSC infusion or localized therapy help repair damaged ovaries?We undertook a careful review of all studies that investigated any strategy using MSCs in either animal models or human studies, to provide evidence that MSCs could improve fertility outcomes.Studies that evaluated 'ovarian reserve'-that is the reproductive potential left within a woman's two ovaries based on number and quality of eggs-were our primary interest.The results showed that different types of MSCs have been tested in attempts to enhance fertility in various contexts.Among these, they improve follicle survival and growth, and are also able to reverse chemotherapy-induced ovarian damage and improve follicle pool survival by boosting ovarian re-growth of blood vessels.Based on recently gathered data, studies in regenerative medicine are yielding encouraging results in terms of restoring fertility.However, work is still needed to optimize techniques and test their safety before they can become available to patients.on multiple factors, such as donor characteristics (age, gender, BMI, ethnicity, pre-existing conditions, and pathologies) (Baer and Geiger, 2012), isolation protocols (different storage temperatures and isolation times), and the flow cytometry protocol applied for cell sorting (Griesche et al., 2010).Another challenge is MSC safety in each particular experimental model.Indeed, risks may be related to the microbiological safety and genetic stability of MSCs after isolation and expansion.There is also potential for adverse events after in vivo use, including concerns about oncogenic safety and control of the host's immune response to MSCs.
Different types of MSCs have been tested in attempts to enhance fertility in various contexts.Protecting the ovarian reserve from aging and gonadotoxic damage and restoring fertility with strategies like in vitro culture and ovarian tissue transplantation remain paramount.

Materials and methods
The aim of this review was to provide evidence of and information on use of MSCs to improve fertility outcomes.We explored MSCs from different animal origins and tissue sources in specific conditions, including: in vitro culture of granulosa cells (GCs), ovarian tissue, and isolated ovarian follicles; ovarian tissue transplantation; and systemic or intraovarian injection after gonadotoxic or age-related follicle pool decline.To this end, we took a systematic approach, reviewing all papers that investigated any of these strategies in either animal models or human studies.The primary outcome was evaluation of the ovarian reserve, with a focus on follicle survival, number, and growth.Secondary outcomes involved analyses of other parameters associated with the follicle pool, such as follicle-related markers like hormones and growth factors, ovarian tissue viability-linked markers like oxidative stress levels, oocyte growth and maturation rates and, of course, pregnancy outcomes.
In line with preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines (Moher et al., 2009), we conducted a PubMed search up to June 2023 using the following keywords for our research: 'mesenchymal stem cells' AND 'ovarian follicles' OR 'ovarian tissue culture' OR 'ovarian follicle culture' OR 'cumulus oocyte complex' (458 records).Only peerreviewed published articles written in English were taken into account.First, all selected studies were imported using Zotero software and duplicates were erased (396 records).Articles were then screened based on their titles (140) and abstracts (121) according to the relevant criteria.Ten more papers were chosen from the references, since they met the same benchmark.After reading the full texts of acquired articles, those fulfilling the required criteria were included (112) (Fig. 2).Ethics approval was not needed because this study did not involve any experimental research.All research data were obtained from published papers.
In all cases, MSCs were found in ovarian stroma surrounding the follicles and never inside follicles, indicating that they are unable to differentiate into GCs or oocytes.All staining methods proved effective and labeled MSCs remained in the ovaries for up to 4 weeks (Fu et al., 2008;Sun et al., 2013;Zarbakhsh et al., 2019;Feng et al., 2020;El-Derany et al., 2021;Pan et al., 2021;Salvatore et al., 2021), 6 weeks (Zhu et al., 2015), and 8 weeks (Ling et al., 2017(Ling et al., , 2019)), irrespective of administration mode.Cell tracking inside the ovaries after systemic administration demonstrated MSC homing capacities toward distant damaged sites (Ling et al., 2017;Besikcioglu et al., 2019;Yang et al., 2019a;El-Derany et al., 2021;Pan et al., 2021;Salvatore et al., 2021).It also showed persistence of MSCs over time, after local ovarian administration proved that they can be retained inside tissue without being cleared by the immune system (Ling et al., 2019;Zarbakhsh et al., 2019).Some authors also investigated their homing capacity toward other distant sites, beyond the ovaries, 24 h after injection, identifying some MSCs in distant organs such as the uterus, spleen, brain, lung, liver, and kidney after both intraovarian (Zhu et al., 2015;Ling et al., 2019;Zhang et al., 2021a) and i.v.injection (Zhu et al., 2015;El-Derany et al., 2021;Salvatore et al., 2021).One research group explored the possibility of increasing homing capacities toward damaged sites using MSCs pretreated by lowintensity pulsed ultrasound (LIPUS) (Ling et al., 2017(Ling et al., , 2022a)).MSC migration was found to increase both in vitro and in vivo in the presence of specific molecular signals, such as stromal cellderived factor 1 (SDF-1), which is enhanced in damaged organs as well as by LIPUS.SDF-1 is a member of the chemokine family, able to drive cell homing through its link to the CXC4 receptor and activation of signals such as phosphatidylinositol 3-kinase/ protein kinase B (PI3K/Akt) (Ling et al., 2022a,b).
Extracellular vesicles include exosomes, macrovesicles and apoptotic bodies, according to their different origin and size.Exosomes are small (40-100 nm diameter) membrane-bound vesicles secreted by cells after invagination of the plasma membrane, before being released into the extracellular space.They can contain proteins, such as cytokines and growth factors, as well as microRNAs (miRNAs) produced by stem cells for paracrine communication purposes, executing comparable functions to their cells of origin in various in vitro and in vivo experimental models.Use of exosomes derived from MSCs has yielded useful information on follicle behavior.Indeed, GCs were able to take in MSC-derived exosomes (Huang et al., 2018), and this ability was apparently maintained in vitro after exposure to chemotherapy (CHT) (Sun et al., 2017;Zhang et al., 2020) and in vivo after systemic administration, followed by homing of extracellular vesicles toward damaged ovaries (Eslami et al., 2023).In studies comparing the impact of exosome versus MSC administration, no difference was encountered in follicle outcomes or restoration of hormone levels (Yang et al., 2020a;Zhang et al., 2020;Eslami et al., 2023).One study did, however, detect slightly higher and longerlasting beneficial effects on the ovarian follicle pool after stem cell injection than exosome administration (Park et al., 2023).
Conditioned medium was mainly used in models of ovarian tissue culture, probably to overcome the difficulties related to different growth rates and metabolic needs of MSCs and ovarian tissue in vitro.Discarded medium is richer than isolated exosomes, as it also contains the entire free protein component constituting the secretome.It may, however, also contain discarded solutes from MSC metabolism, which could hamper the overall effect on follicle survival and growth.The only study directly comparing the impact of MSC-derived medium and MSCs themselves was performed on porcine cumulus-oocyte complexes (COCs) and no significant difference was observed in terms of oocyte maturation or embryo development (Lee, 2021).This demonstrated that, at least in this particular model of in vitro culture, the two methods are equally effective.

Role of miRNAs in ovarian function restoration
Eight papers investigated the role of specific miRNAs as effectors of MSC signaling to GCs (Fu et al., 2017;Sun et al., 2017Sun et al., , 2019;;Ding et al., 2020;Yang et al., 2020a,b;Geng et al., 2022;Qu et al., 2022).miRNAs are small non-coding RNAs, displaying regulatory functions to control fundamental effector proteins in cellular function (Memczak et al., 2013).They are increasingly emerging as key players in a number of pathological conditions, including inflammation and cancer (Aljubran and Nothnick, 2021).Their regulatory impact on specific targets makes them attractive as potential therapeutic tools (Rupaimoole and Slack, 2017).
One study explored the ability of damaged GCs to internalize exosomes and the miRNAs contained within them, and observed an increase in miR-24, miR-106a, miR-19b, and mi-R-25, all related to apoptosis signaling (Sun et al., 2019).A number of studies investigated miRNA content in exosomes derived from human MSCs using large molecular panels and identified miR-17-5p (Ding et al., 2020), miR-664-5p (Sun et al., 2019), miR-369-3p (Geng et al., 2022), and miR-126-3p (Qu et al., 2022) as potential modulators of GC survival and proliferation.MiR-17-5p and miR-126-3p are implicated in regulation of numerous cell activities, including cell cycle progression/arrest (Cloonan et al., 2008;Fang et al., 2015) and PI3K/Akt pathway modulation through interaction with phosphate and tensin homolog (PTEN) (Qu et al., 2022;Geng et al., 2022).Ding et al. (2020) proved that this specific miRNA is able to interact with sirtuin gene family, which are key regulators of mitochondrial activity and cell response to oxidative stress.miR-664-5p and miR-369-3p were found to target and downregulate p53, caspase-3 and hypoxia inducible factor 1 a (HIF-1a), potentially having a beneficial effect on ovarian reserve maintenance in the ovary (Sun et al., 2019;Geng et al., 2022).In all studies, exosome administration resulted in an increase in proliferation and a decrease in apoptosis in CHT-damaged human GCs in vitro, and enhanced follicle survival in a CHT-damaged murine model in vivo.
Expression of specific miRNAs was also modulated in vitro by silencing or enhancing in different experimental models, based on literature evidence of their role in ovarian function.Among others, miR-144-5p was investigated, as its expression is associated with an elevated risk of premature ovarian insufficiency (POI) (Kuang et al., 2014).Its silencing in vitro was found to revive GCs after CHT-induced damage, through PTEN suppression and dysregulation of the PI3K/Akt pathway, confirming its role as a negative effector of follicle maintenance (Yang et al., 2020a).The same in vitro impact was observed by silencing other miRNAs, including miR-146-5p and miR-21-5p (Yang et al., 2020b).As their presence was shown to disrupt follicle growth through PI3K/Akt Stem cell therapy in reproductive medicine | 5 pathway modulation, their silencing yielded better follicle reserve maintenance (Yang et al., 2020b).miR-21, on the other hand, was upregulated by lentiviral transfection (Fu et al., 2017).Its overexpression proved effective at counteracting CHT-induced GC apoptosis.Aging mechanisms of action appeared to be involved in modulating the PTEN and PI3K/Akt pathway.This highlights the crucial role that the PI3K/ Akt pathway plays in follicle maintenance and growth, and also the challenges of fully understanding how its function is governed.

Impact of MSCs on ovarian outcomes in vitro
MSC secretome properties have also been considered as enhancers of follicle survival and growth in in vitro models.Culturing follicles from the primordial stage to fertilizable oocytes is nevertheless a huge challenge (Telfer and Andersen, 2021) and the best approach today involves a multi-step protocol, including: primordial follicle activation and initial growth; follicle development to the antral stage; and oocyte maturation in customized culture conditions to optimize outcomes (McLaughlin et al., 2018).At each step, however, there is significant follicle loss along with follicle growth, and uncertainty about oocyte competence after culture.A number of published studies have investigated the ovarian follicle pool behavior in vitro and speculated whether addition of MSCs could enhance follicle outcomes in terms of survival and growth.
All studies detected a decrease in GC apoptosis in co-culture with MSCs.Some studies also investigated the impact of co-culture on GC proliferation, either by directly demonstrating increased proliferation rates, or observing activation of signaling pathways, such as PI3K/Akt and Hippo, which are known to be involved in GC survival and proliferation (Fu et al., 2017;Huang et al., 2018Huang et al., , 2020;;Hong et al., 2020;Yang et al., 2020a;Li et al., 2021;Park et al., 2021Park et al., , 2023;;Qu et al., 2022).MSC co-culture also appeared to be beneficial for cell hormone function by upregulating markers for steroidogenesis, such as cytochrome P450 19A1 (CYP19A1) and Steroidogenic acute regulatory protein (StAR) (Huang et al., 2018(Huang et al., , 2020;;Park et al., 2021Park et al., , 2023;;Zhang et al., 2021b), and hormone production, such as estradiol (E2), progesterone, anti-M€ ullerian hormone (AMH) and inhibin A and B, in the culture medium (Huang et al., 2018(Huang et al., , 2020;;Yan et al., 2019;Zhang et al., 2021b).MSC co-culture also appeared able to reverse some cellular aging mechanisms, including increased reactive oxygen species (ROS) generation, accumulation of b-galactosidase activity, and elevated methylation of adenosine in mRNA (m6A) in specific genome sites associated with mRNA regulation (Tian et al., 2023).

In vitro culture of ovarian tissue or isolated follicles
Eighteen studies considered use of different sources of MSCs or their derivatives in ovarian tissue culture (Table 2).Selected animal models were rodents (11 studies), ovine (three studies), and pigs (one study), while four studies used human ovarian tissue.The primary outcome was to determine whether MSCs had a positive impact as 'feeder cells' on follicle and/or oocyte culture of: ovine or human ovarian cortical strips (Jia et al., 2017;Hosseini et al., 2019;Arrivabene Neves et al., 2020;Sousa et al., 2021); murine ovaries (Choi et al., 2020;Hong et al., 2020;Buigues et al., 2021a;Cho et al., 2021;Zhang et al., 2021b;Mi et al., 2022;Cao et al., 2023); isolated preantral follicles of murine, ovine, or human origin (Xia et al., 2015;Rajabi et al., 2018;Bezerra et al., 2019;Green et al., 2019;Tomaszewski et al., 2019); and murine or porcine COCs (Maldonado et al., 2018).A positive impact was observed on in vitro follicle populations in all studies using rodent ovarian tissue, showing either increased follicle growth or more follicles with a normal morphology.Conflicting conclusions on the role of MSCs in follicle growth were reached for ovine and human ovarian tissue, with some authors detecting a positive effect (Xia et al., 2015;Bezerra et al., 2019;Hosseini et al., 2019;Sousa et al., 2021) and others not (Jia et al., 2017;Arrivabene Neves et al., 2020).Equally controversial was the impact of MSCs on oocyte outcomes after culture.Indeed, two studies found higher meiotic resumption rates in a murine model (Maldonado et al., 2018;Green et al., 2019), while three others, investigating oocyte growth, meiotic resumption, and maturation rates, did not demonstrate any difference compared to in vitro culture without MSCs (Rajabi et al., 2018;Bezerra et al., 2019;Arrivabene Neves et al., 2020).
These discrepant results may be explained by several factors related to the high variability of the experimental design, not only in the choice of MSC source and ovarian tissue model, but also in the number of cells used for each experiment.Indeed, it is important to note that studies demonstrating a less significant impact of MSCs on ovarian tissue culture are also those using the smallest number of MSCs, for example, 1 � 10 3 cells (Rajabi et al., 2018;Arrivabene Neves et al., 2020), or MSC-conditioned medium (Jia et al., 2017;Bezerra et al., 2019), which may be insufficient to significantly affect oocyte growth and maturation in vitro.Various other markers of ovarian tissue viability have also been assessed, suggesting a positive effect when the MSC secretome is added to in vitro culture.These include: an increase in oocyterelated growth factors like growth differentiation factor 9 (GDF) and bone morphogenic protein 15 (BMP15) (Xia et al., 2015;Rajabi et al., 2018;Hosseini et al., 2019;Lee, 2021); enhanced steroidogenesis (Xia et al., 2015;Rajabi et al., 2018;Green et al., 2019;Hosseini et al., 2019); greater production of growth factors, such as basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor b (TFGb), insulin growth factor 1 (IGF1), vascular endothelial growth factor (VEGF), and epithelial growth factor (EGF) (Tomaszewski et al., 2019;Cho et al., 2021;Lee, 2021); and finally decreased ROS generation in vitro (Bezerra et al., 2019;Lee, 2021).
Ovaries were pretreated with chemotherapeutic drugs (cyclophosphamide and cisplatin) in three studies (Hong et al., 2020;Buigues et al., 2021a;Cao et al., 2023).As in previous reports on CHT-treated GCs, the main outcome was to assess whether MSCs Table 1 Effects of mesenchymal stem cells on granulosa cells in vitro.

Repairing ovarian damage with MSCs
Among the numerous strategies under development to identify an effective treatment for women affected by POI, use of MSCs has been gaining ground over recent years.The goal of stem cell therapy for ovarian rejuvenation is to protect the pool of remaining quiescent follicles still residing in some patients in order to improve their reproductive chances (Polonio et al., 2020).This potential treatment is the result of a number of studies in mice whose ovarian function was damaged by various chemotherapeutic treatments or other toxins, such as ozone.Ovarian injury models have been indeed widely used in research to mimic both infertility and irreversible ovarian failure caused by gonadotoxic drugs depending on dose and mode of administration (Generoso et al., 1971).The impact of MSCs on the remaining ovarian reserve has also been investigated using animal models of natural aging (Polonio et al., 2020).

Ovarian rejuvenation and gonadotoxic damage healing: outcomes and mechanism of action
Outcomes for both ovarian rejuvenation and gonadotoxic damage healing were relatively homogeneous, both showing a positive impact of MSCs on follicle count through regulation of a number of key processes in the ovarian microenvironment.A decrease in apoptosis in all ovarian compartments, including GCs, theca cells and ovarian stroma, was evidenced in the vast majority of the studies.MSCs appear to significantly downregulate various signals responsible for triggering the apoptosis cascade, involving upregulation of antiapoptotic molecules such as B-cell lymphoma 2 (BCL2), survivin (Huang et al., 2020), and NR4A1, which look to be specific to theca cells (Luo et al., 2022), and a shift in the relation between pro-and antiapoptotic signals; for example, the BCL2/BCL2-associated X (BAX) ratio (Zarbakhsh et al., 2019;Geng et al., 2022;Ling et al., 2022b;Luo et al., 2022;Qu et al., 2022;Zhang et al., 2023).This is probably a result of the paracrine signaling of MSCs, which are rich in growth factors like VEGF, HGF, and IGF-1, and able to promote cell survival and proliferation in vitro and in vivo (Uzumcu et al., 2006;Polonio et al., 2020;Chen et al., 2023).
In terms of follicle quality, enhanced follicle growth and improved follicle morphology were often observed.A number of potential mechanisms could be involved in this observation, since follicle growth and function are regulated by a complex interaction of pathways.This includes the ability of follicles to remain quiescent in the ovarian cortex, with oocytes in meiotic arrest, and at the same time ready for recruitment and further growth (Grosbois et al., 2020).One of the main biological functions impacted by the MSC secretome does appear to be primordial follicle activation.Interaction with the PI3K/Akt pathway was indeed demonstrated in several studies, resulting in better follicle growth (Ding et al., 2018;Liu et al., 2019Liu et al., , 2021;;Hong et al., 2020;Huang et al., 2020;Yang et al., 2020a,b;C¸il and Mete, 2021;Deng et al., 2021;El-Derany et al., 2021;Cao et al., 2023).Other cell proliferation signals, like SMADs and c-Jun N-terminal kinase (JNK2) (Bao et al., 2018;Huang et al., 2018;Feng et al., 2020), were also found, as were cell-cell interaction signals such as connexin 43 expression (Sen Halicioglu et al., 2022).Rapid oocyte growth and meiotic resumption require an efficient engine to ensure complete maturation and good quality embryos.This is facilitated by the presence of a large mitochondrial mass and abundance of substrates, including glucose and fatty acids for oxidative phosphorylation (Al-Zubaidi et al., 2021).The impact of MSCs on mitochondrial function in oocytes was also explored in one study, which found a more substantial relevant increase in mitochondrial DNA in the presence of MSCs, a key step allowing further meiotic resumption (Wang et al., 2022).
The effect on mitochondria is part of a more extensive influence that MSCs have on cell function, which has been shown by several authors to exert anti-aging properties (Cacciottola et al., 2021a).Mitochondrial function and control of oxidative stress in cells are among critical factors in cell senescence, and their regulation may explain the rejuvenating effect of stem cell therapy.Moreover, MSCs were found to reverse other specific signaling pathways associated with aging, including upregulation of DNA damage repair mechanisms, namely phosphorylated histone H2AX (cH2AX), breast cancer 1 (BRCA1), poly [ADP-ribose] polymerase 1 (PARP1), and X-ray repair cross complementing 6 (XRCC6) (Huang et al., 2020), as well as cell cycle progression (El-Derany et al., 2021;Tian et al., 2021).One study explored the possibility of enhancing ovarian tissue quality by injecting MSCderived mitochondria into the periovarian space, exhibiting upregulation of gene pathways related to mitochondrial function and energy supply (Zhang et al., 2022a).The experiment did not, however, demonstrate any significant effect on the ovarian reserve, confirming that natural aging is more challenging to reverse than iatrogenic treatments.
Poor follicle quality may also be explained by other less explored mechanisms.Endoplasmic reticulum (ER) stress was investigated in one study as a potential trigger of follicle death, using markers like inositol-requiring enzyme 1a and glucoseregulated protein 78 (Li et al, 2019).MSCs appeared to reverse this cell dysfunction, resulting in restored GC survival.ER stress is caused by the accumulation of unfolded or misfolded proteins caused by various pathological conditions, including oxidative stress and inflammation.It leads to organelle swelling in the form of cytoplasmic vacuoles, which may trigger follicle death by atresia (Hay et al., 1976).While massive ER dysfunction owing to complete loss of calcium homeostasis and membrane lipid damage is very hard to reverse, moderate ER stress levels may allow oocyte maturation (Lin et al., 2012;Takahashi et al., 2019).It is therefore important to act upon this cell mechanism to ensure the development of normal and fertilizable oocytes, having completed maturation, especially after in vitro growth (Harada et al., 2015;McLaughlin et al., 2018).

MSCs for ovarian tissue transplantation
Twelve studies on ovarian tissue transplantation were identified and included in the review (Table 5).The majority of studies on human ovarian tissue were published by two research groups in China (Xia et al., 2015;Cheng et al., 2022) and two research groups in Europe, namely the UCL Gynecology Research Unit in Brussels, Belgium (Manavella et al., 2018(Manavella et al., , 2019;;Cacciottola et al., 2020Cacciottola et al., , 2021b,c) ,c) and the Reproductive Medicine Research Group in Valencia, Spain (Herraiz et al., 2018a;Buigues et al., 2021a,b).Two studies involved autologous ovarian transplantation in murine models (Damous et al., 2018;Yang et al., 2020b), where MSCs were either injected into the ovaries or systemically, taking advantage of their homing capacities, or grafted locally with the help of biocompatible scaffolds (fibrin or Matrigel).
Ovarian tissue transplantation outcomes are currently limited by massive follicle death occurring shortly after grafting because of both hypoxia-mediated apoptosis and non-physiological follicle activation (Dolmans et al., 2021a).Transplantation approaches using MSCs to boost early graft revascularization may well address this issue in a clinical setting and enhance transplantation results.All patients would potentially benefit, but especially those with lower chances of fertility restoration using this technique.This includes subjects showing signs of an already depleted follicle reserve in their cryopreserved ovarian cortex, or with a damaged pelvis caused by repeated surgery or previous pelvic irradiation, making it unable to properly host ovarian grafts (Dolmans et al., 2021b).
Table 5 Effects of mesenchymal stem cells in models of ovarian tissue transplantation.

Ongoing clinical applications
Use of MSCs to increase the likelihood of pregnancy in postmenopausal patients has already been recorded in a clinical context, with publication of case reports and small case series.The first live births were obtained by intraovarian BM-MSC injection by Edessy et al. (2016) in 1 out of 10 treated patients with POI (26-33 years), by Gabr et al. (2016) in 1 out of 30 treated patients with POI (18-40 years), and by Gupta et al. (2018) in one patient aged 45 years.Preliminary results on DOR subjects were published by Herraiz et al. (2018b) and the study is still ongoing.Seventeen patients with DOR (<39 years) undergoing intraarterial catheterization for MSC mobilization using GCS-F were included.An improvement in ovarian function was observed in around 80% of patients within 4 weeks.There were increased numbers of antral follicles, particularly in the infused ovary, and also retrieved oocytes, with a significant drop in cancelation rates after controlled ovarian stimulation (Herraiz et al., 2018b).Five pregnancies were obtained, three of which resulted in a healthy baby at the time of publication.The same group (Herraiz et al.) recruited 20 patients with POI (<39 years of age) for a new study investigating both intra-arterial catheterization and stem cell mobilization in peripheral blood using granulocyte-colony stimulating factor.The second technique relying on peripheral blood, which is much less invasive than the original approach, appears to have a systemic effect on both ovaries after stem cell injection, thanks to their homing ability to distant damaged sites, as previously suggested by several animal studies (Polonio et al., 2020).Only preliminary data have been published so far, reporting menses recovery in around 40% of patients and one pregnancy to date (Polonio et al., 2020).Clinical application of sources other than BM-MSCs for fertility restoration in patients with POI have also been published recently.One study reported menses recovery in four out of nine patients (29-39 years) treated by intraovarian injection of AT-MSCs (Mashayekhi et al., 2021).Another study reported menses recovery and increased antral follicle count in 4 out of 15 POI patients treated with autologous Men-MSCs (Zafardoust et al., 2023).In terms of fertility restoration, four live births were obtained after treating 61 patients with POI (<35 years old) with UC-MSCs (Yan et al., 2020).
All published studies have reported live birth rates of around 10% in patients with POI.These may be considered comparable to spontaneous pregnancy rates of around 5-10% in women with POI in the first few years after their diagnosis, owing to spontaneous but temporary ovarian reactivation (Fraison et al., 2019).While these results are promising and corroborated by sound evidence from animal studies, ovarian rejuvenation with MSCs has not yet delivered the expected results.Future studies need to focus on ways of enhancing fertility in these patients (Rosario and Anderson, 2021).One way may be maximizing the effect of stem cell use by choosing the best conditions, in terms of cell type, concentration, and administration mode, in order to boost their biological impact on the dormant ovarian reserve.Another way may be advancing our understanding of the pathophysiology of POI in order to be able to select the subgroup of patients with the best chance of benefiting from this technique.

Discussion/conclusions
The present article offers a comprehensive overview of possible future applications of MSCs in reproductive medicine.MSCs from different sources, including fetal and adult tissues, have been tested in different conditions, as have their derivatives (exosomes and the secretome contained in culture medium).The majority of studies consider follicle recovery after CHT exposure, with numerous in vitro studies on GCs and in vivo studies in murine models.All of them investigate the potentially beneficial impacts of MSCs on the follicle pool during or immediately after gonadotoxic damage, providing more in-depth knowledge of the ability of GCs to recover, as well as the dynamics governing follicle death and abnormal activation after injury.This model does not, however, represent the clinical situation of young women diagnosed with DOR or POI caused by gonadotoxic therapy years prior.This could be the reason for the discrepancy between outcomes of preclinical studies, which clearly show significant protection of the follicle pool mediated by MSCs, and results from the first clinical trials, in which the MSC effect appears to be much more modest.Further studies are needed to better understand the impact of MSCs on the follicle pool, particularly on oocyte quality, which may be the limiting factor responsible for the disappointing results.
Other promising clinical applications involving MSCs are emerging from the literature, in our quest to enhance fertility outcomes.Ovarian tissue co-culture with MSCs, used as feeder cells to improve follicle survival, growth, and oocyte competence, may serve to propel the ovarian tissue in vitro culture technique toward legitimate clinical application.Similarly, MSCs have also proved effective at boosting revascularization of the grafting site in the context of ovarian tissue transplantation.Indeed, data from preclinical studies using human ovarian tissue xenografting models appear robust and reproducible, suggesting a possible role for MSCs in counteracting large-scale ovarian follicle pool loss after grafting, which is still one of the main limiting factors of the technique.
To sum up, all gathered data on the one hand show that regenerative medicine techniques are quickly gaining ground among the innovative techniques being developed for future clinical application in the field of reproductive medicine.On the other hand, there is still a lot of work to do before MSCs can be safely and effectively used to improve follicle outcomes in different clinical applications.We are moving in the right direction but need to delve deeper to advance our fundamental understanding of these multipotent cells.

Figure 1 .
Figure 1.Sources of mesenchymal stem cells and their capacity for differentiation.Fetal (placenta, umbilical cord, amniotic fluid) and adult (skin, adipose tissue, bone marrow) sources of pluripotent mesenchymal stem cells are able to grow in vitro and differentiate into different tissues, including bone, muscle, cartilage, adipose tissue, vessels, skin, and nerves.Unique populations of MSCs by their multilineage differentiation capacity, facility to grow as adherent cells in standard culture conditions, and ability to express specific marker profiles.

Figure 2 .
Figure 2. PRISMA flow diagram of literature search to June 2023.Literature search methodology for publications investigating use of mesenchymal stem cells in reproductive medicine to enhance follicle outcomes.PRISMA, preferred reporting items for systematic reviews and meta-analyses.
in reproductive medicine | 7

Table 2
Impact of mesenchymal stem cells on ovarian tissue in vitro.

Table 3
Studies using mesenchymal stem cells in models of ovarian aging.

Table 4
Effects of mesenchymal stem cells in ovarian damage models.